Arg20突变对敬钊缨毛蛛毒素-V钠通道活性的影响  

Effects of Arg20 Mutation on Sodium Channels Activity of JZTX-V

作　　者：曾雄智[1] 邓梅春[1] 皮建辉[2] 全妙华[2] 王贤纯[1] 梁宋平[1] 

机构地区：[1]湖南师范大学蛋白质化学与发育生物学教育部重点实验室,长沙410081 [2]怀化学院生物工程系,怀化418008

出　　处：《生物工程学报》2008年第7期1228-1232,共5页Chinese Journal of Biotechnology

基　　金：国家973项目(No.2006CB708508)与863项目(No.2006AA02Z141);国家自然科学基金重点项目与面上项目(Nos.30430170,30670640,30500146);湖南省自然科学基金项目(No.07JJ3072);湖南省教育厅资助科研项目(No.06C503)~~

摘　　要：敬钊缨毛蛛毒素-V(Jingzhaotoxin-V,JZTX-V)是从敬钊缨毛蛛粗毒中纯化到的一种新型河豚毒素不敏感型钠通道抑制剂,为了深入研究该毒素的结构与功能关系,应用芴甲氧羰基(Fmoc)固相多肽化学合成方法合成了用丙氨酸(Ala)替代JZTX-V第20位精氨酸残基的突变体R20A-JZTX-V,合成线性多肽经反相高效液相色谱分离纯化后进行谷胱甘肽氧化复性。复性产物分别用基质辅助激光解析飞行时间质谱(MALDI-TOF/TOF MS)进行分子量的鉴定,用膜片钳电生理方法进行电压门控钠通道抑制活性分析。研究结果表明,Arg20被Ala取代后,R20A-JZTX-V对大鼠背根神经节细胞(DRG)膜上表达的河豚毒素敏感型(TTX-S)钠通道的抑制活性与天然JZTX-V相当,提示Arg20与JZTX-V对TTX-S钠通道的抑制活性无关或关系不大;而R20A-JZTX-V对TTX-R钠通道的抑制活性却比天然JZTX-V下降了约18.3倍,说明Arg20是与JZTX-V对河豚毒素不敏感型(TTX-R)钠通道抑制活性相关的关键活性残基之一,推测R20A-JZTX-V活性降低的原因是用Ala替代Arg20后改变了JZTX-V与TTX-R型钠通道的作用位点。Jingzhaotoxin-V（JZTX-V） isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in adult rat dorsal root ganglion（DRG） neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structure-function relationship of the toxin, a mutant of JZTX-V in which Arg20 was substituted by Ala, was synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography and oxidatively refolded under the optimal conditions. The refolded product was analyzed by matrix-assisted laser desorptiord ionization time-of-flight mass spectrometry（MALDI-TOF MS） and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of R20A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V, suggesting that Arg20 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast, the prohibitive level of R20A-JZTX-V on TTX-R sodium channels is reduced by at last 18.3 times, indicating that Arg20 is a key amino acid residue relative to the bioactivity of JZTX-V. It is presumed that the decrease in activity of R20A-JZTX-V is due to the changes of the property in the binding site in TTX-R sodium channels.

关 键 词：敬钊缨毛蛛毒素-V固相多肽合成 突变体 氧化复性 膜片钳 

分 类 号：Q51[生物学—生物化学] TQ453.01[化学工程—农药化工]